The difficulties in generating effective T-cell based vaccines and immunotherapies against chronic infections with viruses such as HCV and HIV highlight our limited understanding of what defines a successful versus an inadequate T cell response. Studies in human infection will be critical to better guide new approaches for inducing or improving T cell responses and recent technological advances give opportunity to overcome previous boundaries in assessing the T cell response. One important and understudied aspect of the T cell response is that T cells at the site of infection are and have to be qualitatively different from those circulating in the blood. For obvious reasons, studies on human tissue-resident T cells are limited to material either removed for clinical indications or very small tisse samples that can be removed without significant risk for the individual. The challenge is to obtain material that allows meaningful comparisons and to generate complex information from typically very small numbers of cells that are available for analysis. HCV infection is one of the most common chronic viral infections in man and has two key features that make it an excellent system for studies of immune protection and immune failure in humans: 1) HCV infection can be both chronic and self-limiting and 2) tissue from HCV+ individuals can be routinely obtained through surgery, biopsies and fine needle aspiration. We will utilize these two key features of HCV infection in conjunction with novel approaches for analyzing small numbers of cells of interest to define critical features of T-cells directly from the site of infection that do and do ot control the virus. Our hypothesis is that in chronic infection T cells with specific signatures of dysfunction, exhaustion and dysregulation will enrich in the liver, whereas in resolved infection fully formed memory T cells with be detectable in blood and liver. In aim 1 we will use multiplex gene expression analysis to compare HCV-specific T cells in chronic and resolved infection, using cells from both the liver and the blood. Aim 2 will significantly increase the potential scop of the analytical approach established in aim 1 by adapting it for material from fine needle aspirations. These can be performed electively in well-defined clinical situations, allowing to extend the study of intrahepatic T cells to subjects with acute infection or other clinical timepoints of immunological interest in which so far only T cells from the blood have been analyzed. With completion of the studies proposed in this exploratory/developmental application we will have gained novel insights into critical aspects of protective immunity and immune failure in humans and will also have laid the foundation for prospective studies of immunological perturbations in humans.